Therapeutic agent

ABSTRACT

Agent for the depletion of an unwanted protein population from the plasma of a subject, which agent comprises a plurality of ligands covalently co-linked so as to form a complex with a plurality of the proteins in the presence thereof, wherein at least two of the ligands are the same or different and are capable of being bound by ligand binding sites present on the proteins, wherein the agent is a non-proteinaceous agent other than a D-proline of the formula  
                 
wherein 
 
R is  
                 
the group 
     R 1  is hydrogen or halogen;    X is —(CH 2 ) n —; —CH(R 2 )(CH 2 ) n —; —CH 2 O(CH 2 ) n —; —CH 2 NH—; benzyl, —C(R 2 )═CH—; —CH 2 CH(OH)—; or thiazol-2,5-diyl;    Y is —S—S—; —(CH 2 ) n —; —O—; —NH—; —N(R 2 ); —CH═CH—; —NHC(O)NH—; —N(R 2 )C(O)N(R 2 )—; —N[CH 2 C 6 H 3 (OCH 3 ) 2 ]—; —N(CH 2 C 6 H 5 )—; —N(CH 2 C 6 H 5 )C(O)N(CH 2 C 6 H 5 )—; —N(alkoxyalkyl)-; N(cycloalkyl-methyl)-; 2,6-pyridyl; 2,5-furanyl; 2,5-thienyl; 1,2-cyclohexyl; 1,3-cyclohexyl; 1,4-cyclohexyl; 1,2-naphthyl; 1,4-naphthyl; 1,5-naphthyl; 1,6-naphthyl; biphenylen; or 1,2-phenylen, 1,3-phenylen and 1,4-phenylen, wherein the phenylen groups are optionally substituted by 1-4 substituents, selected from halogen, lower alkyl, lower alkoxy, hydroxy, carboxy, —COO-lower alkyl, nitrilo, 5-tetrazol, (2-carboxylic acid pyrrolidin-1-yl)-2-oxo-ethoxy, N-hydroxycarbamimidoyl, 5-oxo[1,2,4]oxadiazolyl, 2-oxo-[1,2,3,5]oxathiadiazolyl, 5-thioxo[1,2,4]oxadiazolyl and 5-tert-butylsulfanyl-[1,2,4]oxadiazolyl;    X′ is —(CH 2 ) n —; —(CH 2 ) n CH(R 2 )—; —(CH 2 ) n OCH 2 —; —NHCH 2 —; benzyl, —CH═C(R 2 )—; —CH(OH)CH 2 ; or thiazol-2,5-diyl;    R 2  is lower alkyl, lower alkoxy or benzyl and n is 0-3, or a pharmaceutically acceptable salt or mono- or diester thereof.

The present invention relates to an agent for the depletion of anunwanted protein population from the plasma of a subject, use of theagent for the preparation of a therapeutic composition, and a method fortreatment using the agent.

BACKGROUND TO THE INVENTION

Proteins in the blood plasma, in the extracellular matrix of thetissues, and in cells are essential for all vital physiologicalfunctions. However, many different proteins also contribute to disease.The underlying pathogenetic mechanisms include overproduction of normalproteins with corresponding excessive effects, the production ofabnormal proteins with damaging effects, and the incidental subversionof normal protein function leading to damaging effects duringintercurrent pathological processes, such as inflammation and microbialinfection. There is therefore a need for elimination of a variety ofnormal or abnormal proteins from the body to provide treatment for manyhuman diseases. Plasma proteins that contribute to pathogenesis ofdisease include cytokines, lipoproteins, autoantibodies, components ofthe complement and coagulation pathways, amyloidogenic proteinsincluding monoclonal immunoglobulin light chains, transthyretin,lysozyme and β₂-microglobulin, acute phase proteins in particular serumamyloid A protein (SAA), and pentraxins such as serum amyloid Pcomponent (SAP). All these different proteins, produced by differentcells, are potentially attractive targets for therapeutic elimination invarious diseases. However, there are few effective methods forselectively lowering the circulating concentration of specific proteinsand those approaches that are available, or have been attempted, arecomplex, difficult and subject to many extremely challenging problems.

Cytokines are protein hormones produced by and acting on a variety ofdifferent cell types, and are critically important mediators of hostdefence, immunological and inflammatory responses, but theiroverproduction in some diseases contributes to serious pathology,morbidity and mortality. Antibodies and recombinant binding proteinshave lately been used successfully for targeting one particularcytokine, tumor necrosis factor (TNF), and TNF blockade istherapeutically beneficial in rheumatoid arthritis and Crohn's disease,but there are few effective methods for reducing damaging high levels ofother cytokines.

Pathogenic overproduction of other normal proteins, such as acute phaseplasma proteins, can be reduced by suppressing the activity of anunderlying primary disease, but, except in the case of treatable chronicinfection, this is usually extremely difficult to achieve. There is nocure for chronic idiopathic inflammatory diseases, such as rheumatoidarthritis or Crohn's disease, or for most malignant neoplasms. Treatmentof these diseases and suppression of their activity require regimensincluding highly toxic anti-inflammatory, cytotoxic andimmunosuppressive drugs, and frequently also surgery and/orradiotherapy.

Production of abnormal proteins, whether inherited or acquired as acomplication of another primary disease, is also extremely difficult tocontrol. For example, the variant transthyretin and variant fibrinogenmolecules responsible for forms of hereditary systemic amyloidosis, canbe eliminated from the plasma only by liver transplantation. No suchapproach is possible with many other hereditary diseases caused bypathogenic variant proteins. Acquired production of abnormal andpathogenic proteins, as in monoclonal gammopathies, can be treated onlywith powerful cytotoxic drugs or bone marrow transplantation. Theseprocedures incur universal morbidity, and even mortality rates of up50%, without being uniformly successful.

Another approach has been to remove damaging proteins by extracorporealabsorption, passing blood over more or less selective solid phase mediato remove the target protein. This can make a useful contribution toremoval of the high concentrations of pro-atherogenic low densitylipoprotein in familial hypercholesterolaemia. It has also beenattempted, so far unsuccessfully, for removal of two differentamyloidogenic proteins: β₂-microglobulin in patients on chronichaemodialysis for end stage renal failure, and variant transthyretin inpatients with familial amyloid polyneuropathy.

In cases where particular proteins produce their pathogenic effects bybinding to specific ligand structures in vivo, a possible approach totherapy is the development of drugs to inhibit such binding. Forexample, the present inventor has proposed such an approach to thetreatment of amyloidosis and Alzheimer's disease, targeting thepathogenic binding of serum amyloid P component to amyloid fibrils byadministration of low molecular weight molecules that block such binding(1-5) U.S. Pat. No. 6,126,918). The specific binding of SAP toparticular ligands containing anionic groups, including carboxylates andphosphates is known (6-10). In the complex of SAP with deoxyadenosinemonophosphate (dAMP), there is a dAMP molecule in the calcium-dependentbinding site of each protomer in the homopentameric SAP molecule (10).As a result of base stacking, dependent on hydrogen bonding betweenpairs of dAMP molecules, pairs of SAP pentamers loaded with dAMPassemble face to face to form a decameric protein-ligand complex (10).

The known low molecular weight ligands of SAP, such asphosphoethanolamine and methyl4,6-O-(1-carboxyethylidene)-β-D-galactopyranoside (MOβDG), are onlybound with modest affinity of about millimolar. This is poor compared tothe typical nanomolar affinities of most drug-protein interactions, andsuggests that such compounds would be unlikely to be effective asinhibitors of the pathogenic binding of SAP to its ligands in vivo.There remains a need to provide an effective method for the targeteddepletion of an unwanted plasma protein population.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the present invention provides an agentfor the depletion of an unwanted protein population from the plasma of asubject, which agent comprises a plurality of ligands covalentlyco-linked so as to form a complex with a plurality of the proteins inthe presence thereof, wherein at least two of the ligands are the sameor different and are capable of being bound by ligand binding sitespresent on the proteins, wherein the agent is a non-proteinaceous agent.Agents disclosed in EP-A-0915088 do not form part of this aspect of theinvention. EP-A-0915088 discloses D-prolines of the formula

wherein

-   R is    the group-   R¹ is hydrogen or halogen;-   X is —(CH₂)_(n)—; —CH(R²)(CH₂)_(n)—; —CH₂O(CH₂)_(n)—; —CH₂NH—;    benzyl, —C(R²)═CH—; —CH₂CH(OH)—; or thiazol-2,5-diyl;-   Y is —S—S—; —(CH₂)_(n)—; —O—; —NH—; —N(R²)—; —CH═CH—; —NHC(O)NH—;    —N(R²)C(O)N(R²)—; —N[CH₂C₆H₃(OCH₃)₂]—; —N(CH₂C₆H₅)—;    —N(CH₂C₆H₅)C(O)N(CH₂C₆H₅)—; —N(alkoxyalkyl)-; N(cycloalkyl-methyl)-;    2,6-pyridyl; 2,5-furanyl; 2,5-thienyl; 1,2-cyclohexyl;    1,3-cyclohexyl; 1,4-cyclohexyl; 1,2-naphthyl; 1,4-naphthyl;    1,5-naphthyl; 1,6-naphthyl; biphenylen; or 1,2-phenylen,    1,3-phenylen and 1,4-phenylen, wherein the phenylen groups are    optionally substituted by 1-4 substituents, selected from halogen,    lower alkyl, lower alkoxy, hydroxy, carboxy, —COO-lower alkyl,    nitrilo, 5-tetrazol, (2-carboxylic acid    pyrrolidin-1-yl)-2-oxo-ethoxy, N-hydroxycarbamimidoyl,    5-oxo[1,2,4]oxadiazolyl, 2-oxo-[1,2,3,5]oxathiadiazolyl,    5-thioxo[1,2,4]oxadiazolyl and    5-tert-butylsulfanyl-[1,2,4]oxadiazolyl;-   X′ is —(CH₂)_(n)—; —(CH₂)_(n)CH(R²)—; —(CH₂)_(n)OCH₂—; —NHCH₂—;    benzyl, —CH═C(R²)—; —CH(OH)CH₂; or thiazol-2,5-diyl;-   R² is lower alkyl, lower alkoxy or benzyl and-   n is 0-3,    and pharmaceutically acceptable salts or mono- or diesters thereof.

Surprisingly, it has been found that agents according to the presentinvention are dramatically potent in vivo in not only inhibiting ligandbinding but also depleting the target protein from the circulation bycausing it to be rapidly cleared. The present invention provides ageneric method for identification and/or creation of drug molecules thatare specifically bound by individual target proteins and then engage thenormal, exquisitely sensitive, capacity of the body to recognise anddestroy autologous proteins that have undergone changes in conformationor assembly.

Accordingly, there is provided use of a non-proteinaceous agent for thepreparation of a composition for the depletion of an unwanted proteinpopulation from the plasma of a subject, which agent comprises aplurality of ligands covalently co-linked so as to form a complex with aplurality of the proteins in the presence thereof, wherein at least twoof the ligands are the same or different and are capable of being boundby ligand binding sites present on the proteins.

The normal physiological role of each protein depends critically on itsappropriate molecular conformation and/or assembly, and the body haspowerful mechanisms to detect and destroy proteins that are damaged,aggregated or misfolded. The purpose of the present invention is tospecifically target individual proteins and cause them to be identifiedby the body's own physiological mechanisms as requiring prompt clearanceand destruction. In order to achieve this effect the inventionadvantageously uses palindromic agents that aggregate the proteins asdimers or higher order aggregates.

The exact structure of the agent of the present invention will bedependent upon the protein or proteins of the unwanted proteinpopulation targeted according to the invention. In a preferredembodiment, the unwanted population consists essentially of a singleprotein species which will bear one or, in some cases, more than oneligand binding site. Many proteins are specifically equipped, as part oftheir normal function, to bind particular low molecular weight ligands.In the simple case where the single protein species has a single ligandbinding site, each ligand in the therapeutic agent would be selected tobe capable of being bound in that ligand binding site. The ligand couldbe selected from the ligands known to be bound by that binding site,ligands predicted to be bound by that site perhaps on the basis ofstructural information available on the binding site such as X-raycrystallographic information, or structural analogues thereof. Fortarget proteins without known low molecular weight ligands, suitablecompounds can be identified by high throughput screening of chemicallibraries and/or structure based molecular design. The affinity of eachindividual ligand-protein binding site interaction does not need to beparticularly high provided that the ligand is specific for each targetprotein. It is possible that a dissociation constant of up to 10millimolar would suffice. However, it is preferred that the dissociationconstant is no more than 1 millimolar, more preferably less than 100micromolar, most preferably less than 10 micromolar. The affinity ispreferably about micromolar or higher. Micromolar affinity has beenfound to be sufficient in the case of SAP, although the highest possibleaffinity is clearly desirable.

In the agents of the present invention, although the ligands may bedirectly linked together by a covalent bond, the ligands are preferablycovalently co-linked by a linker. This enables the ligands to besufficiently spatially separated whereby a plurality of target proteinsmay be bound to the agent without one protein hindering the binding ofthe other protein or proteins. The exact structure of the linker is notcritical although it is typically preferred not to include reactivegroups. The linker may comprise a linear or branched hydrocarbylenewhich may have one or more of its carbon atoms optionally substituted bya heteroatom. The linker may have a chain length in the range 2 to 20atoms. Useful chain length and chemical composition may be determinedempirically depending on the proteins with which the agent is to becomplexed. Where the agent has two ligands, the linker is typicallylinear; a preferred general structure is ligand-linker-ligand. This isconveniently denoted a “palindrome” for the purposes of the presentapplication. Although other structures involving three, four or moreligands with an appropriate branched chain linker are also contemplatedwhere three, four or more target proteins could form a complex.

In a further embodiment, at least two of the ligands in the agent aredifferent from one another and are capable of binding differentproteins. In this embodiment, the unwanted protein population consistsessentially of two protein species or more. In this way, two differenttypes of protein can be targeted for depletion. It is possible to use aprotein known to be rapidly depleted, such as SAP, to enhance theclearance of another target protein by designing the agent with oneligand capable of being bound by SAP and having a second differentligand for targeting the different target protein for clearance with theSAP.

The target protein may be a normal molecule or an abnormal, variant,molecule. The ligand binding site of the agent according to theinvention may be from a cytokine, a lipoprotein, an autoantibody, anacute phase protein, an amyloidogenic protein or a component of thecomplement or coagulation pathway. Where the ligand binding site is froman acute phase protein, this may comprise SAA.

Where the ligand binding site is from an amyloidogenic protein, this maycomprise a monoclonal immunoglobulin light chain, β₂-microglobulin,transthyretin or lysozyme.

Where the ligand binding site is from lysozyme, at least one of theligands comprises a disaccharide or oligosaccharide analogue containingat least N-acetyl muramic acid linked via its C1 atom to the C4 atom of,for example, N-acetyl glucosamine, with the O atom of the 1,4βglycosidic linkage replaced by a carbon or other non-O atom.

In the case of SAP, one class of agents comprises the D-prolines offormula set out above, preferably(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid or a pharmaceutically acceptable salt ormono- or diester thereof.

The agent may be used to deplete unwanted protein populations from theplasma of human or animal subjects. At appropriate molar ratios of agentto target protein, the agent cross links pairs of protein molecules toproduce complexes that are recognised in the body as abnormal and arethen promptly cleared and catabolised. This leads to substantial orcomplete depletion of the target protein and confers therapeuticbenefit.

Pharmaceutical compositions may be formulated comprising an agentaccording to the present invention optionally incorporating apharmaceutically-acceptable excipient, diluent or carrier. Thepharmaceutical compositions may be in the form of a prodrug comprisingthe agent or a derivative thereof which becomes active only whenmetabolised by the recipient. The exact nature and quantities of thecomponents of such pharmaceutical compositions may be determinedempirically and will depend in part upon the route of administration ofthe composition. Routes of administration to recipients include oral,buccal, sublingual, by inhalation, topical (including ophthalmic),rectal, vaginal, nasal and parenteral (including intravenous,intra-arterial, intra-muscular, subcutaneous and intra-articular) Forconvenience of use, dosages according to the present invention arepreferably administered orally but this will depend on the actual drugand its bioavailability. A typical dosage will be 50 to 500 mg per dayorally or by continuous intravenous infusion, or intermittentsubcutaneous injection, for example of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid.

BRIEF DESCRIPTION OF THE INVENTION

The present invention will now be described in further details by way ofexample only, with reference to the following Examples and to theaccompanying drawings, in which:

FIG. 1 shows the three dimensional X-ray crystal structure of SAPcomplexed with an agent of the present invention;

FIG. 2A shows a detailed view from FIG. 1 and FIG. 2B shows a detailedalternative representation of part of FIG. 1;

FIG. 3 shows the effect of an agent of the present invention on SAP inmice in vivo;

FIG. 4 shows the effect of infusion of an agent of the invention onplasma SAP values in patients with systemic amyloidosis;

FIG. 5 shows the effect of the agent over six hours using ¹²³I-SAPscintigraphy;

FIG. 6 shows the effect of the agent up to 48 hours using ¹²³I-SAPscintigraphy;

FIG. 7 shows the plasma concentration of the agent and its effect on SAPduring intravenous infusion; and

FIG. 8 shows the effect of subcutaneous injection of the agent inpatients with systemic amyloidosis.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES

Serum Amyloid P Component and(R)-1-[6-(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicAcid

A method for screening and testing inhibitors of serum amyloid Pcomponent (SAP) binding to amyloid fibrils in vitro was devised and usedin collaboration with F Hoffmann-La Roche Ltd to identify a suitablelead molecule for drug development. A library of candidate compounds wasscreened accordingly. The subsequent collaborative medicinal chemistryprogramme led to synthesis of a family of dicarboxylic acid, pyrrolidonering containing molecules, of which the most studied is(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid. This molecule and related compounds (EP-A-0915088) are modestlypotent inhibitors of SAP binding to amyloid fibrils in vitro, with IC₅₀values around 0.5-1.0 μM. However they are all more potent inhibitorsthan the original lead compound, which was1-(3-Mercapto-2-methyl-1-oxopropyl)-D-proline, containing just a singleD-proline ring and carboxylate.

SAP is a pentamer with 5 identical non-covalently associated protomerseach bearing a single calcium dependent ligand binding site on oneplanar face, the binding (B) face, of the molecule (9). In the absenceof calcium, human SAP forms stable decameric dimers, probably via A-faceto A-face interactions (11). In the presence of calcium, isolated humanSAP rapidly aggregates and precipitates, as a result of molecularlattice formation due to binding of the exposed carboxylate of theGlu167 residue on one SAP molecule by the calcium dependent ligandbinding site of another SAP molecule (12,13). This autoaggregation ofSAP is inhibited by other ligands to which SAP binds, and all thepresent inhibitors of SAP binding to amyloid fibrils were active in thisrespect. However we show here that the greater potency of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid and related compounds, as inhibitors of SAP binding to amyloidfibrils in vitro, results from their capacity to cross link pairs of SAPmolecules. The palindromic structure of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid enables it to not only block the ligand binding sites on individualSAP protomers, but could also cross link pairs of pentameric SAPmolecules to form B-B face to face decameric dimers. Gel filtrationanalysis of mixtures of isolated SAP with(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid, at ratios between equimolar and 100 fold molar excess of drug(molecular weight 340 Da) to SAP protomers (25,462 Da), show that allthe SAP is decameric (Table 1). However at 128 fold or greater molarexcess of drug, all the SAP elutes in a volume corresponding to itssingle pentameric form, because each binding site is then occupied by adifferent individual(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid molecule, preventing cross linking and dimerisation. TABLE 1Molecular assembly of isolated pure SAP in the presence of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid in vitro Drug:protein CalciumElution molar ratio present Protein volume (ml) Molecular assembly Nodrug − CRP 12.28 Pentamer No drug + CRP 12.81 Pentamer 1:1 + CRP 12.58Pentamer 10:1  + CRP 11.96 Pentamer No drug − SAP 10.93 Decamer 1:1 +SAP 10.97 Decamer 10:1  + SAP 10.97 Decamer 128:1  + SAP 12.55 Pentamer

C-reactive protein (CRP) is a pentraxin closely related to SAP. Samplesof isolated pure human SAP or CRP were mixed with(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid at the molar ratios shown, with respect to pentraxin protomer, inphysiological ionic strength Tris buffered saline pH 8.0, and analysedby size exclusion chromatography precisely as described previously (14).The mixtures and the column eluants contained either no calcium ions or2 mM calcium, and the concentrations of drug appropriate to maintain themolar ratios indicated above. In the absence of a specific ligand towhich it can bind, purified human SAP is insoluble in the presence ofcalcium and elutes as high molecular weight aggregates in the voidvolume of the column (14). However in the absence of calcium and ligand,isolated SAP alone forms stable decamers, providing an excellent markerfor the elution volume of this molecular assembly (14). Human CRP is astable pentamer either in the presence or absence of calcium and thusprovides a robust marker for the elution position of that molecular form(14).

The molecular model of two native pentameric SAP molecules cross linkedby binding between them of the palindromic(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid molecule, is confirmed by the three dimensional X-ray crystalstructure of the complex of SAP with the drug in the presence ofcalcium, as shown in representative views below (FIGS. 1 & 2). Thesefindings are consistent with the published observations on the SAP-dAMPcomplex (10) and the chromatographic analysis (Table 1). FIG. 1 shows athree dimensional X-ray crystal structure of SAP complexed with(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid. In this Figure there is a side view showing two pentamericdisc-like SAP molecules (9,10) edge on, cross linked by five(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid molecules, the terminal D-proline residues of each lying in thecalcium-dependent ligand binding pocket of the apposed protomers. FIG.2. shows a three dimensional X-ray crystal structure of SAP complexedwith(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid. Detailed views are set out in FIGS. 2A and 2B showing twodifferent representations of the ligand binding residues in thecalcium-dependent ligand binding pockets of two apposed protomers ofdifferent SAP molecules, and the electron density and/or the molecularstructure of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid lying between them.

Experimental Studies of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicAcid In Vivo

In vivo studies of 1-(3-Mercapto-2-methyl-1-oxopropyl)-D-proline, theoriginal lead compound, show that it both inhibits binding of SAP toexperimentally induced amyloid deposits in mice and dissociates SAP thatis already bound in the deposits. Studies with(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid confirmed that it is more active in these respects than1-(3-Mercapto-2-methyl-1-oxopropyl)-D-proline. The potency of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid in vivo was initially considered to be consistent with its lowerIC₅₀ value in vitro. However the decameric assembly of pentameric SAPmolecules, induced by the cross linking capacity of the palindromicdrug, is an abnormal molecular configuration which, according to thepresent invention, will be recognised in vivo and promptly cleared fromthe circulation. This action of the drug, leading to massive depletionof SAP from the plasma, makes a critical contribution to removal of SAPfrom the amyloid deposits because the SAP in amyloid is derived from,and in equilibrium with, the plasma SAP pool.

FIG. 3 shows as follows the effects of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid on SAP in mice in vivo. Upper panel:groups of 10 age, sex and weight matched mice with experimentallyinduced reactive systemic AA amyloidosis were given a single loadingdose of ¹²⁵I-labelled human SAP on day −1, and received implantedosmotic minipumps delivering the doses shown of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (designated on the figures as “desapin” for convenience). Clearancefrom the amyloid deposits, catabolism and excretion of the human SAPtracer were monitored globally by whole body counting of the mice(left), and the total amount of mouse SAP in the amyloidotic organs wasdetermined after killing the animals on day 5 (right). Middle panel,left: 4 groups each of 10 age, sex and weight matched mice withexperimentally induced reactive systemic AA amyloidosis received anintravenous injection of ¹²⁵I-labelled pure human or mouse SAP at timezero and were then bled after 15 minutes and at the other times shown.Immediately after the 15 minute bleed, two groups, one that had receivedhuman SAP and the other mouse SAP, received a single intraperitonealinjection of 5 mg of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (desapin). Tracer remaining in the circulation is expressed as themean (SD) percentage of the amount at the 15 minute time point for eachgroup. Middle panel, right: groups each of 10 age, sex and weightmatched mice with experimentally induced reactive systemic AAamyloidosis received an intravenous injection of ¹²⁵I-labelled purehuman or mouse SAP at time zero and were then bled after 15 minutes andat the other times shown. In one of each of the pairs of groupsreceiving mouse or human SAP respectively, the tracer had been mixedwith(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (desapin) and incubated in vitro before injection into the animals.Tracer remaining in the circulation is expressed as mean (SD) totalradioactivity per gram of blood in each group. Bottom panel: effect ofadministration of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (desapin) in the drinking water at the concentrations shown, oncirculating human SAP values in human SAP transgenic mice. Mice ofapproximately 20 g body weight consume about 3 ml water per day.

(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was not metabolised in mice and was very rapidly excreted,predominantly in the urine, with a small amount in the bile. However,even intermittent intraperitoneal or subcutaneous injection of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid inhibited uptake of radiolabelled human SAP tracer intoexperimentally induced mouse AA amyloid deposits. When(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was administered by continuous infusion for 5 days via anindwelling osmotic pump, it dissociated both radiolabelled human SAPtracer, with which the amyloid deposits had previously been loaded, andall the endogenous mouse SAP in the deposits (FIG. 3). Even 50 μg/kg/dayof(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid significantly dissociated human SAP from the deposits (not shown),but 1.5 mg/kg/day was required to dissociate any endogenous mouse SAPsignificantly, and there was a clear dose response effect up to 15mg/kg/day, which removed all the mouse SAP (FIG. 3). A singleintraperitoneal injection of 5 mg of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid accelerated the plasma clearance of radiolabelled human SAP tracerin normal mice, but had no significant effect on the clearance ofradiolabelled mouse SAP (FIG. 3). However, when either radiolabelledmouse or human SAP was preincubated with (R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid in vitro, beforeintravenous injection into normal mice, both tracers were extremelyrapidly cleared (FIG. 3). TheSAP-(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid complex is evidently recognised as abnormal in vivo and swiftlyremoved from the circulation, but is apparently formed and/or clearedless efficiently in vivo with mouse SAP compared to human SAP.Remarkably, despite very limited oral bioavailability, administration of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid in the drinking water to human SAP transgenic mice, which have meanplasma values of about 80 mg/l of human SAP, rapidly depleted theircirculating human SAP (FIG. 3). The human SAP transgenic animals do nothave any circulating mouse SAP, but neither oral administration, norintermittent injection or continuous infusion, of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid to normal wild type mice, caused depletion of their circulatingSAP.

Clinical Studies of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicAcid in Human Amyloidosis

FIG. 4 shows the effect of intravenous infusion of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid on plasma SAP values in patients with systemic amyloidosis.Patients with different forms of systemic amyloidosis, and with varyingamyloid loads received the doses of the drug shown for a period of 48 h.Circulating SAP values were measured by electroimmunoassay (24) insamples taken during and after the infusion, at the times shown. Eachline represents a different individual patient.

(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was administered for 48 h by intravenous infusion to 8 patientswith systemic amyloidosis (7 with AL and one with AA type), one patientwith minor localised AL amyloidosis, and one who is a carrier of theamyloidogenic Ala60 transthyretin variant but has no clinicalamyloidosis. There was dramatic, rapid, and consistent depletion ofcirculating SAP in all subjects. In initial studies with slow continuousinfusion, the SAP concentration started to fall when about 2 mg of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid had been given, and in subsequent studies this amount was thereforeinjected as a bolus at the outset, followed by infusion at the ratesshown (FIG. 4). At an infusion rate of 0.1 mg/kg/day, SAP depletion wasslower and less complete, but at 0.25 mg/kg/day and all higher rates upto a maximum of 6 mg/kg/day, SAP was almost completely cleared from theplasma by the end of the infusion, regardless of the amyloid load (FIG.4). However, after drug infusion had ceased, the plasma SAPconcentration rapidly returned to normal in the individuals with littleor no amyloid, whereas this recovery was markedly delayed in subjectswith significant amyloidosis. In the one patient with a very heavyamyloid load, the plasma SAP concentration remained below 25% of itsinitial value 20 days after the infusion. Most of the daily productionof SAP, which is about 50-100 mg per day (15), was evidentlydistributing into the amyloid deposits before becoming available toreplete the plasma pool. This is very strong indirect evidence that evensuch a brief infusion of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid had substantially depleted the amyloid-associated SAP.

FIG. 5 shows whole body ¹²³I-SAP scintigraphy before and 6 h afterstarting infusion of(R)-1-[6-(R)-2-Carboxy-pyrrolidine-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid. This patient, with a modest load of AL amyloid in the spleen andkidneys, had notable blood pool background of tracer in the heart andcirculation before administration of the drug. At 6 h the blood poolbackground is completely absent and the liver, which is the only site ofcatabolism of SAP in vivo (16,17), has taken up the tracer whilst theintensity of SAP signal from the amyloidotic spleen and kidneys issignificantly reduced (organ counts not shown).

FIG. 6 shows whole body ¹²³I-SAP scintigraphy before and 24 h and 48 hafter starting in fusion of(R)-1-[6-(R)-2-Carboxy-pyrrolidine-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid in a second patient. This patient, with modest load of AL amyloidin the spleen and kidneys, had notable blood pool background of tracerin the heart and circulation before administration of the drug. At 24 hand 48 h the blood pool background is completely absent and the liver,which is the only site of catabolism of SAP in vivo (16,17), has takenup the tracer and catabolic products are being excreted in the urine, asshown by the bladder signal. The intensity of SAP signal from theamyloidotic spleen and kidneys is significantly reduced: at 24 h spleencounts are 85% of time 0, kidneys 77%; at 48 h, spleen 54%, kidneys 50%.

Direct evidence for depletion of SAP from amyloid in the organs and forthe mechanism of action of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was obtained by quantitative whole body scintigraphy using ¹²³I-SAPas a tracer. Each patient received a standard dose of ¹²³I-SAP 24 hbefore the(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid infusion started and was scanned immediately before treatment toprovide a baseline image and values for localisation of tracer to theamyloid deposits. They were then scanned at intervals thereafter, up tothe end of the 48 h infusion.(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid caused dramatic clearance of tracer from the plasma, exactlyparalleling both in the scintigraphic images and in counting of bloodsamples, the SAP depletion monitored by immunoassay of the serum. By 6 hafter starting treatment, and persisting thereafter, the blood poolsignal virtually disappeared (FIGS. 5, 6), and there was strikingaccumulation of tracer in the liver, identifying this organ as the siteto which(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid caused clearance of the circulating SAP. At the same time there wasa marked decrease in the retention of tracer in amyloid depositselsewhere, exemplified by the spleen and kidneys, in contrast to theusual situation in control untreated amyloidosis patients (Table 2; FIG.6). TABLE 2(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid rapidly clears circulating SAP to the liver and depletesSAP from visceral amyloid deposits ¹²³I-SAP retention [mean (SD) %]Liver Spleen Time after tracer Time after tracer injection No. ofinjection No. of Treatment 24 h 48 h patients 24 h 48 h patients None100 78 (8) 12 100 86 (25) 14 Drug 100 125 (20) 7 100 54 (18) 7 P <0.0001 P = 0.008

All patients had systemic AL amyloidosis and received a standardintravenous tracer dose of 1231-SAP at time zero. After whole bodyquantitative scintigraphic imaging at 24 h, uptake in liver and spleenwere taken as 100% for each individual. Intravenous infusion of the drug(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was then started and scintigraphy with organ counting was repeatedat 48 h. The controls received no treatment. Significance of differencesbetween the two groups was sought by t-test.

It has previously been demonstrated in mice that the liver, andspecifically the hepatocyte, is the only significant site of clearanceand catabolism of both mouse and human SAP in vivo (16). Furthermore,asialo-human SAP is instantly cleared by the liver in man, via thehepatocyte asialoglycoprotein receptor, and this process has been imagedusing ¹²³I-asialo SAP (17).(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid evidently triggers similarly rapid hepatic uptake of SAP in vivo,leading to virtually total depletion of circulating SAP. This promotesredistribution of SAP from the tissues to the plasma and supplements theeffect of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid as a competitive inhibitor of SAP binding to amyloid fibrils,thereby leading to highly efficient removal of SAP from amyloiddeposits.

FIG. 7 shows plasma concentration of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid and of SAP during 48 h infusion. Two individual patients withsystemic amyloidosis and moderate amyloid loads received(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (desapin) by intravenous infusion at the steady rates shown.Circulating SAP concentrations started to fall almost immediately andhad halved when the concentrations of drug and SAP protomer wereequimolar.

The potency of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid in depleting circulating SAP was remarkable, with SAP valuesfalling when the concentration ratio of drug to SAP pentamer in theplasma approached equimolar (FIG. 7). In vitro gel filtration studiesshowed that even this very low concentration of drug is sufficient togenerate some SAP dimers (Table 3), that is pairs of pentameric SAPmolecules cross linked by(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid molecules to form the decameric assembly seen in the crystalstructure (FIGS. 1, 2). This is evidently the species recognised asabnormal and promptly cleared by the liver. TABLE 3 Molecular assemblyof SAP in whole human serum in the presence of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid in vitro Drug:protein CalciumElution molar ratio present Protein volume (ml) Molecular assembly Nodrug − CRP 12.0-12.5 Pentamer No drug + CRP 12.0-12.5 Pentamer 1:1 + CRP12.0-12.5 Pentamer 10:1  + CRP 12.0-12.5 Pentamer 100:1  + CRP 12.0-12.5Pentamer No drug − SAP 10.5-11.5 Decamer No drug + SAP  7.0-8.0 High MWaggregates 1:1 + SAP  7.0-8.0 & High MW aggregates 10.0-10.5 Decamer10:1  + SAP  7.0-8.0 & High MW aggregates 10.0-10.5 Decamer 100:1  + SAP10.0-10.5 Decamer

Aliquots of a single pool of whole normal human serum, containing SAPand CRP, were mixed with(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid at the molar ratios shown, with respect to pentraxin protomer, andanalysed by size exclusion chromatography precisely as describedpreviously (14). The mixtures and the column eluants contained either nocalcium ions or 2 mM calcium, and the concentrations of drug appropriateto maintain the molar ratios indicated above. SAP in whole serum is astable soluble pentameric assembly of protomers, stabilised by thepresence of the normal high concentration of serum albumin. However asSAP is separated from serum albumin during gel filtration with a calciumcontaining eluant, the SAP autoaggregates and elutes entirely as highmolecular weight polymers in the void volume of the column. This isbecause, in the absence of a specific ligand to which it can bind,isolated human SAP is insoluble in the presence of calcium (14). Howeverin the absence of calcium, where no ligand binding or autoaggregationcan occur, SAP forms stable decamers, providing an excellent marker forthe elution volume of this molecular assembly (14). Human CRP is astable pentamer either in the presence or absence of calcium and thusprovides a robust marker for the elution position of that molecular form(14). Even at an equimolar concentration of the drug, some of the SAPwas solubilised as stable drug-decamer complexes, and at 100 fold molarexcess of drug, all the SAP was in this form.

The potency of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid was confirmed by the observation that subcutaneous injection ofjust 0.25 mg/kg/day, given in 12 hourly divided doses, had the sameeffect on plasma SAP (FIG. 8) as intravenous infusion of the drug.

FIG. 8 shows the effect of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid administration by subcutaneous injection on circulating SAP valesand visceral organ retention of ¹²³I-SAP tracer. The doses of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid (desapin) shown were given by 12 hourly subcutaneous injections totwo individual patients with systemic amyloidosis, and with small andmoderate amyloid loads respectively. The disappearance of circulatingSAP was effectively the same as during intravenous infusion of the drug,and in the patient illustrated in the right panel, who had received atracer dose of ¹²³I-SAP, there was accumulation of SAP in the liver andaccelerated clearance from the spleen.

CONCLUSION

(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid specifically targets SAP in vivo, through the specific ligandbinding capacity of SAP, but additionally, as a consequence of thedrug's palindromic structure, according to the present invention, itcauses aggregation of native pentameric SAP molecules into decamericdrug SAP complexes that are then promptly cleared by the liver. Thisnovel effect was not intended during development of(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid as an inhibitor of SAP binding to amyloid fibrils, and was neithersought nor expected. It represents a well documented example of thepresent invention and it dramatically enhances the dissociation of SAPfrom amyloid deposits, which is the therapeutic objective inamyloidosis. Such targeted pharmacological depletion of a circulatingplasma protein represents a novel, previously undescribed mechanism ofdrug action, with broad applicability to many different proteins anddisease processes.

Transthyretin

Transthyretin (TTR), formerly known as prealbumin, is a normal plasmaprotein produced in the liver and also by the choroid plexus in thebrain. It transports both thyroid hormone (thyroxine andtri-iodothyronine) and retinol binding protein (RBP) in the plasma.Although these are presumably important physiological functions, micewith targeted deletion of the TTR gene and complete absence of TTR growand develop normally, are fertile and have no abnormal phenotype. Thisis probably because retinol (vitamin A) can be supplied adequatelywithout the need for carriage of RBP by TTR, and because there isanother plasma protein that specifically binds thyroid hormone,thyroxine binding globulin (TBG). Indeed TBG binds thyroxine with muchgreater affinity than TTR and is evidently more important in regulatinghormonal availability and function.

Normal wild type TTR is inherently amyloidogenic and at post mortemexamination almost all individuals over the age of 80 years havemicroscopic deposits of TTR amyloid in the heart, blood vessel walls,choroid plexus or elsewhere. In some subjects the deposits are much moresubstantial and may cause clinical problems, a condition known as senilesystemic amyloidosis. Importantly, there are over 60 mutations in thehuman TTR gene, encoding single amino acid substitutions in variant TTRproteins, that cause the very serious and generally fatal form ofautosomal dominant hereditary systemic amyloidosis known as familialamyloid polyneuropathy. This condition is variably penetrant but whenexpressed it is invariably fatal with onset in early to middle adultlife and inexorable progression of various combinations of peripheraland autonomic neuropathy, cardiac, renal, vascular and ocularinvolvement, leading to death within 5-15 years. There are thousands ofaffected kindreds throughout the world. The only effective treatment isliver transplantation, which replaces the source of the amyloidogenicvariant plasma TTR with a source of normal wild type TTR, and over 1000such operations have been performed world wide since the procedure wasintroduced in 1991 (18,19). Once the pathogenic variant TTR is replaced,deposition of amyloid is halted and the existing deposits regress withclinical benefit, provided the patient's condition is not already tooadvanced. However liver transplantation is clearly a major and dangerousprocedure, especially in these patients with multiple organ systemdamage by amyloid, and there is an insoluble shortage of donor organs.Furthermore, satisfactory outcomes have largely been confined topatients with the most common amyloidogenic TTR variant, Met30Val. Ithas lately become apparent that in patients with other amyloidogenicmutations of the TTR gene, liver transplantation does not haltprogression of amyloid deposition, especially in the heart, and despitethe presence only of wild type TTR in the plasma the outcome remainspoor. There is thus a pressing need for new therapeutic approaches.

One such approach is the use of drugs that are bound with high affinityby TTR, in order to stabilise the native fold of the TTR molecule andprevent the unfolding and subsequent refolding into the pathogenicamyloid cross-β structure that underlies amyloid fibrillogenesis. Lowmolecular weight molecules suitable for use as drugs of this type havebeen developed and effectively inhibit formation of amyloid fibrils byisolated TTR in vitro (20). Furthermore some of these compounds havebeen shown to be specifically bound with high affinity, not just byisolated TTR, but also by TTR within the environment of whole plasma(21). This is critically important because drugs in general are oftenbound by multiple plasma proteins, especially albumin which comprises57% by weight of all plasma proteins. Also, in particular, drugs boundby TTR in the binding pocket that specifically recognises thyroidhormone, may be bound by TBG as well.

According to the present invention, palindromic drugs that are bound byTTR in vivo, and can then cross link TTR molecules, aggregating them andthereby marking them for prompt clearance from the circulation, aresuitable for use in depleting TTR and therefore treating and preventingTTR amyloidosis. The ligand head groups of such drugs may be compounds9-11 in Purkey et al (21), as set out below. The linker joining them maybe a series of methylene groups as used in the SAP depleting compound,(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid, detailed above, or another aliphatic and/or aromatic chain ofsuitable length and other properties.

When these ligands are bound by TTR, the ring bearing the carboxylatesits deeply in the cavity within the TTR tetramer, and the ring bearingthe halogen atoms is peripheral and exposed on the surface of theligand-TTR complex. The attachment point for the correct bond or linkeris preferably a point on the aromatic ring bearing the halogen orhalogen-containing substituents. A more preferred attachment point forthe linker, forming compounds according to the present invention, is thering carbon lying between the two ring carbons that have halogensattached, or a carbon of the ring distal to the carboxylate bearing ringin the middle example above.

Lysozyme

Lysozyme is a glycanase enzyme (EC 3.2.1.17) widely distributed inanimals, plants and lower organisms. It specifically cleaves theglycosidic bond between the C-1 of N-acetylmuramic acid and the C-4 ofN-acetylglucosamine in bacterial peptidoglycan, and is thus a1,4-β-N-acetylmuramidase. In man lysozyme is secreted in saliva, tearsand other external secretions, is the major secreted product ofmacrophages and is also produced by hepatocytes and the Paneth cells ofthe intestine. The functions of lysozyme in man are not well understood,although by virtue of its capacity to digest the cell walls ofsusceptible bacteria it may contribute to host defence. It is alsohighly cationic and becomes tightly associated with anionicglycosaminglycans and is concentrated in cartilage. The first mutationsto be discovered in the human lysozyme gene are associated with a formof hereditary systemic amyloidosis for which there is no effectivetreatment (22). The variant lysozyme molecules are amyloidogenic becausethe single residue substitutions they contain render them less stablethan wild type lysozyme (23). They spontaneously unfold underphysiological conditions, populating partly unfolded states that have ahigh propensity to refold and aggregate in the abnormal, pathogenic,amyloid cross-β fibrillar configuration (23).

According to the present invention, palindromic drugs that are bound bylysozyme in vivo, and can then cross link lysozyme molecules,aggregating them and thereby marking them for prompt clearance from thecirculation, are suitable for use in depleting lysozyme and thereforetreating and preventing lysozyme amyloidosis. The ligand head groups ofsuch drugs are stereochemically related to lysozyme substrates but arebound and not cleaved by the enzyme. For example, a disaccharide oroligosaccharide analogue containing at least N-acetyl muramic acidlinked via its C1 atom to the C4 atom of N-acetyl glucosamine, with theO atom of the 1,4β glycosidic linkage replaced by a carbon or othernon-O atom, is bound by lysozyme but cannot be hydrolysed. Appropriatesubstituents, such as fluorine or nitrogen, on the critical C atom, thatreplaces the hydrolysable O atom of the glycosidic linkage, can enableimportant hydrogen bonding contributed by the replaced O atom to beretained. The linker joining the ligands may be a series of methylenegroups as used in(R)-1-[6-(R)-2-Carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylicacid, or another aliphatic and/or aromatic chain of suitable length andother properties. The preferred attachment points for the linker arepositions 2 (N-acetyl) or 6 (OH) on any of the pyranose rings, as thesepositions point to the exterior in the oligosaccharide-lysozyme complex.

β₂-Microglobulin

β₂-microglobulin is a single chain protein of Mr 11,815 that isnon-covalently associated with all class I MHC molecules on the surfaceof cells. It is produced at the rate of about 200 mg per day in man andis cleared and catabolised exclusively in the kidney. Plasma levels ofβ₂-microglobulin therefore rise in renal failure and neitherhaemodialysis nor peritoneal dialysis are effective in clearingβ₂-microglobulin. In patients with end stage renal failure on dialysis,the plasma concentration of β₂-microglobulin reaches 50-70 mg/l,compared to the normal of about 2 mg/l, and after about 5-7 years ofdialysis most such individuals suffer from deposition ofβ₂-microglobulin amyloid, especially around bones and joints. This leadsto serious morbidity and mortality among the approximately 1 millionpatients who are on long term dialysis world wide. The only effectivetreatment is renal transplantation, which provides the only possibleroute for efficient removal of β₂-microglobulin, but transplantation isavailable only for a small minority of end stage renal failure patients.

According to the present invention, palindromic drugs that are bound byβ₂-microglobulin in vivo, and can then cross link β₂-microglobulinmolecules, aggregating them and thereby marking them for promptclearance from the circulation, are suitable for use in depletingβ₂-microglobulin and therefore treating and preventing β₂-microglobulinamyloidosis. Specific ligands for β₂-microglobulin, bound withreasonable affinity, can be identified by high throughput screening ofchemical libraries. β₂-microglobulin shares substantial sequencehomology and its tertiary fold with immunoglobulin light chains, and itscrystal structure is known at atomic resolution, enabling and greatlyfacilitating rational molecular design.

Amyloid Fibril Precursor Proteins in General

It is well established that in all forms of amyloidosis in which it ispossible to eliminate the supply of amyloid fibril precursor proteins,deposition of new amyloid ceases and existing deposits either stabiliseor regress leading to clinical benefit. This has been detailed in theexamples above but it applies to all types of amyloidosis, includingthose associated with Alzheimer's disease, the transmissible spongiformencephalopathies including bovine spongiform encephalopathy and variantCreutzfeldt-Jakob disease, and type 2, maturity onset diabetes mellitus.

Drugs according the present invention, specific for each particularamyloid fibril precursor protein, are applicable to all these and otheramyloid related diseases. Furthermore, heterobifunctional drugsaccording to the present invention, in which one species of ligand headis bound specifically by SAP and the other by the target amyloidogenicprotein, are especially advantageous. They promote efficient removal ofthe target protein itself, they do so with enhanced efficiency byengaging the powerful clearance mechanism that operates on aggregatedSAP, and they also simultaneously eliminate SAP itself. Since SAPcontributes to the pathogenesis of all types of amyloidosis, thesimultaneous removal of both the amyloidogenic fibril proteins and theSAP has synergistic therapeutic benefit.

Autoantibodies

The acquired immune system of the body has the capacity to make highlyspecific, high affinity, antibodies against essentially any molecule.Molecules that induce antibody formation are called antigens, and thesubmolecular region that is actually recognised and bound by an antibodyis called an epitope. Normally antibodies are formed predominantly inresponse to foreign antigens from outside the body and are of centralimportance in host defence against microbial and parasitic infection.Apart from low grade responses that contribute to clearance of damagedautologous constituents, and other responses that are involved in immuneregulation, the immune system is tolerant towards the body's ownmolecules and does not produce autoantibodies against them. However inautoimmune diseases, tolerance is broken and aberrant autoantibodies areproduced that bind to self constituents and lead to inflammation, celldeath, and tissue damage. There are many different forms of autoimmunedisease, depending on the precise specificity of the autoantibodies andwhether they are directed against organ or tissue specific autoantigens,or are non-organ specific. However in most autoimmune diseases thespecificity of the pathogenic autoantibodies and the identities of theautoantigens and many of their key epitopes are known.

According to the present invention, palindromic drugs that are boundspecifically by particular pathogenic autoantibodies in vivo, and canthen cross link them, producing aggregation leading to prompt clearancefrom the circulation, are suitable for use in depleting theseautoantibodies and therefore treating autoimmune diseases.

Examples of pathogenic autoantibodies and the diseases they cause, thatare suitable for treatment according to the present invention include:anti-DNA antibodies in systemic lupus erythematosus (SLE) and relatedcollagen diseases, anti-immunoglobulin antibodies (rheumatoid factors)in rheumatoid arthritis and related idiopathic arthritides,anti-phospholipid antibodies in SLE and related collagen diseases, allthe organ specific autoantibodies in all the organ specific autoimmunediseases including those that affect the endocrine organs, the liver,gut, skin, muscle, central and peripheral nervous systems, eyes, ears,heart, blood vessels, lungs and other viscera, and all the autoimmunediseases affecting the red cells, white cells and platelets of the bloodand their precursors in the bone marrow. In these and all the otherautoimmune diseases, the present invention provides for creation ofpalindromic drug molecules comprising the relevant specific epitope orepitopes as the ligand head groups, joined by a linker structure.

REFERENCES

-   1. Hind, C. R. K., Collins, P. M., Caspi, D., Baltz, M. L. and    Pepys, M. B. (1984) Specific chemical dissociation of fibrillar and    non-fibrillar components of amyloid deposits. Lancet, ii: 376-378.-   2. Tennent, G. A., Lovat, L. B. and Pepys, M. B. (1995) Serum    amyloid P component prevents proteolysis of the amyloid fibrils of    Alzheimer's disease and systemic amyloidosis. Proc. Natl. Acad. Sci.    USA, 92: 4299-4303.-   3. Pepys, M. B., Tennent, G. A., Booth, D. R., Bellotti, V.,    Lovat, L. B., Tan, S. Y., Persey, M. R., Hutchinson, W. L.,    Booth, S. E., Madhoo, S., Soutar, A. K., Hawkins, P. N., Van    Zyl-Smit, R., Campistol, J. M., Fraser, P. E., Radford, S. E.,    Robinson, C. V., Sunde, M., Serpell, L. C. and    Blake, C. C. F. (1996) Molecular mechanisms of fibrillogenesis and    the protective role of amyloid P component: two possible avenues for    therapy. In: The nature and origin of amyloid fibrils, (Bock, G. R.    and Goode, J. A., eds.), Wiley, Chichester (Ciba Foundation    Symposium 199), pp. 73-89.-   4. Pepys, M. B., Booth, D. R., Hutchinson, W. L., Gallimore, J. R.,    Collins, P. M. and Hohenester, E. (1997) Amyloid P component. A    critical review. Amyloid: Int. J. Exp. Clin. Invest., 4: 274-295.-   5. Pepys, M. B. (1999) The Lumleian Lecture. C-reactive protein and    amyloidosis: from proteins to drugs? In: Horizons in Medicine, Vol.    10 (Williams, G., eds.), Royal College of Physicians, London, pp.    397-414.-   6. Pepys, M. B., Dash, A. C., Munn, E. A., Feinstein, A., Skinner,    M., Cohen, A. S., Gewurz, H., Osmand, A. P. and    Painter, R. H. (1977) Isolation of amyloid P component (protein AP)    from normal serum as a calcium-dependent binding protein. Lancet, i:    1029-1031.-   7. Pontet, M., Engler, R. and Jayle, M. F. (1978) One step    preparation of both human C-reactive protein and Clt. Fed. Eur.    Biol. Soc. Lett., 88: 172-178.-   8. Hind, C. R. K., Collins, P. M., Renn, D., Cook, R. B., Caspi, D.,    Baltz, M. L. and Pepys, M. B. (1984) Binding specificity of serum    amyloid P component for the pyruvate acetal of galactose. J. Exp.    Med., 159: 1058-1069.-   9. Emsley, J., White, H. E., O'Hara, B. P., Oliva, G., Srinivasan,    N., Tickle, I. J., Blundell, T. L., Pepys, M. B. and    Wood, S. P. (1994) Structure of pentameric human serum amyloid P    component. Nature, 367: 338-345.-   10. Hohenester, E., Hutchinson, W. L., Pepys, M. B. and    Wood, S. P. (1997) Crystal structure of a decameric complex of human    serum amyloid P component with bound dAMP. J. Mol. Biol., 269:    570-578.-   11. Ashton, A. W., Boehm, M. K., Gallimore, J. R., Pepys, M. B. and    Perkins, S. J. (1997) Pentameric and decameric structures in    solution of serum amyloid P component by X-ray and neutron    scattering and molecular modelling analyses. J. Mol. Biol., 272:    408-422.-   12. Baltz, M. L., de Beer, F. C., Feinstein, A. and    Pepys, M. B. (1982) Calcium-dependent aggregation of human serum    amyloid P component. Biochim. Biophys. Acta, 701: 229-236.-   13. Booth, D. R., Gallimore, J. R., Hutchinson, W. L., Hohenester,    E., Thompson, D., Wood, S. and Pepys, M. B. (1999) Analysis of    autoaggregation and ligand binding sites of serum amyloid P    component by in vitro mutagenesis. In: Amyloid and Amyloidosis 1998,    (Kyle, R. A. and Gertz, M. A., eds.), Parthenon Publishing, Pearl    River, New York, pp. 23-25.-   14. Hutchinson, W. L., Hohenester, E. and Pepys, M. B. (2000) Human    serum amyloid P component is a single uncomplexed pentamer in whole    serum. Mol. Med., 6: 482-493.-   15. Hawkins, P. N., Wootton, R. and Pepys, M. B. (1990) Metabolic    studies of radioiodinated serum amyloid P component in normal    subjects and patients with systemic amyloidosis. J. Clin. Invest.,    86: 1862-1869.-   16. Hutchinson, W. L., Noble, G. E., Hawkins, P. N. and    Pepys, M. B. (1994) The pentraxins, C-reactive protein and serum    amyloid P component, are cleared and catabolized by hepatocytes in    vivo. J. Clin. Invest., 94: 1390-1396.-   17. Pepys, M. B., Rademacher, T. W., Amatayakul-Chantler, S.,    Williams, P., Noble, G. E., Hutchinson, W. L., Hawkins, P. N.,    Nelson, S. R., Gallimore, J. R., Herbert, J., Hutton, T. and    Dwek, R. A. (1994) Human serum amyloid P component is an invariant    constituent of amyloid deposits and has a uniquely homogeneous    glycostructure. Proc. Natl. Acad. Sci. USA, 91: 5602-5606.-   18. Holmgren, G., Steen, L., Ekstedt, J., Groth, C.-G., Ericzon,    B.-G., Eriksson, S., Andersen, O., Karlberg, I., Norden, G.,    Nakazato, M., Hawkins, P., Richardson, S. and Pepys, M. (1991)    Biochemical effect of liver transplantation in two Swedish patients    with familial amyloidotic polyneuropathy (FAP-met³⁰). Clin. Genet.,    40: 242-246.-   19. Holmgren, G., Ericzon, B.-G., Groth, C.-G., Steen, L., Suhr, O.,    Andersen, O., Wallin, B. G., Seymour, A., Richardson, S.,    Hawkins, P. N. and Pepys, M. B. (1993) Clinical improvement and    amyloid regression after liver transplantation in hereditary    transthyretin amyloidosis. Lancet, 341: 1113-1116.-   20. Klabunde, T., Petrassi, H. M., Oza, V. B., Raman, P.,    Kelly, J. W. and Sacchettini, J. C. (2000) Rational design of potent    human transthyretin amyloid disease inhibitors. Nature Struct.    Biol., 7: 312-321.-   21. Purkey, H. E., Dorrell, M. I. and Kelly, J. W. (2001) Evaluating    the binding selectivity of transthyretin amyloid fibril inhibitors    in blood plasma. Proc. Natl. Acad. Sci. USA, 98: 5566-5571.-   22. Pepys, M. B., Hawkins, P. N., Booth, D. R., Vigushin, D. M.,    Tennent, G. A., Soutar, A. K., Totty, N., Nguyen, O., Blake, C. C.    F., Terry, C. J., Feest, T. G., Zalin, A. M. and Hsuan, J. J. (1993)    Human lysozyme gene mutations cause hereditary systemic amyloidosis.    Nature, 362: 553-557.-   23. Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V.,    Hutchinson, W. L., Fraser, P. E., Hawkins, P. N., Dobson, C. M.,    Radford, S. E., Blake, C. C. F. and Pepys, M. B. (1997) Instability,    unfolding and aggregation of human lysozyme variants underlying    amyloid fibrillogenesis. Nature, 385: 787-793.-   24. Nelson, S. R., Tennent, G. A., Sethi, D., Gower, P. E.,    Ballardie, F. W., Amatayakul-Chantler, S. and Pepys, M. B. (1991)    Serum amyloid P component in chronic renal failure and dialysis.    Clin. Chim. Acta, 200: 191-200.

1-17. (canceled)
 18. A method for the depletion of a disease-associatedprotein population from the plasma of a subject in need of suchtreatment, which comprises: (a) administering to the subject atherapeutically effective amount of a non-proteinaceous agent, whichagent comprises a plurality of ligands covalently co-linked to permitcomplexation with a plurality of the disease-associated proteins in thepresence thereof, wherein at least two of the ligands are the same ordifferent and are capable of being bound by ligand binding sites presenton the proteins, (b) binding of at least two of the ligands by theligand binding sites of the proteins in the plasma; (c) forming therebya complex between the agent and a plurality of the proteins, wherein thecomplex is abnormal to the subject; and (d) causing the complex to beidentified by the physiological mechanisms of the subject and clearedfrom the plasma; and (e) monitoring the clearance of thedisease-associated protein population from the subject's plasma.
 19. Themethod according to claim 18, wherein the agent is a D-proline of theformula

wherein R is the group

R¹ is hydrogen or halogen; X is —(CH₂)_(n)—; —CH(R²)(CH₂)_(n)—;—CH₂O(CH₂)_(n)—; —CH₂NH—; benzyl, —C(R²═CH—; —CH₂CH(OH)—; orthiazol-2,5-diyl; Y is —S—S—; —(CH₂)_(n)—; —O—; —NH—; —N(R²)—; —CH═CH—;—NHC(O)NH—; —N(R²)C(O)N(R²)—; —N[CH₂C₆H₃(OCH₃)₂]—; —N(CH₂C₆H₅)—;—N(CH₂C₆H₅)C(O)N(CH₂C₆H₅)—; —N(alkoxyalkyl)-; N(cycloalkyl-methyl)-;2,6-pyridyl; 2,5-furanyl; 2,5-thienyl; 1,2-cyclohexyl; 1,3-cyclohexyl;1,4-cyclohexyl; 1,2-naphthyl; 1,4-naphthyl; 1,5-naphthyl; 1,6-naphthyl;biphenylen; or 1,2-phenylen, 1,3-phenylen and 1,4-phenylen, wherein thephenylen groups are optionally substituted by 1-4 substituents, selectedfrom halogen, lower alkyl, lower alkoxy, hydroxy, carboxy, —COO-loweralkyl, nitrilo, 5-tetrazol, (2-carboxylic acidpyrrolidin-1-yl)-2-oxo-ethoxy, N-hydroxycarbamimidoyl,5-oxo[1,2,4]oxadiazolyl, 2-oxo-[1,2,3,5]oxathiadiazolyl,5-thioxo[1,2,4]oxadiazolyl and 5-tert-butylsulfanyl-[1,2,4]oxadiazolyl;X′ is —(CH2)_(n)—; —(CH₂)_(n)CH(R²)—; —(CH₂)_(n), OCH₂—; —NHCH₂—;benzyl, —CH═C(R²)—; —CH(OH)CH²; or thiazol-2,5-diyl; R² is lower alkyl,lower alkoxy or benzyl and n is 0-3, or a pharmaceutically acceptablesalt or mono- or diester thereof.
 20. The method according to claim 19,wherein the D-proline is(R)-1-[6-(R)-2-Carboxypyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid or a pharmaceutically acceptable salt ormono- or diester thereof.
 21. The method according to claim 18, whereinthe ligands are covalently co-linked in the agent by a linker.
 22. Themethod according to claim 21, wherein the linker comprises a linear orbranched hydrocarbylene in which one or more of the carbon atoms thereofis optionally substituted by a heteroatom.
 23. The method according toclaim 18, wherein the agent has two ligands.
 24. A method for thedepletion of a disease-associated protein population from the plasma ofa subject in need of such treatment, which comprises administering tothe subject a therapeutically effective amount of a non-proteinaceousagent, which agent has the general structure Ligand-linker-Ligand and iscapable of forming a complex with a plurality of the disease-associatedproteins in the presence thereof, wherein the ligands are the same ordifferent and are capable of being bound by ligand binding sites presenton the proteins; and monitoring the clearance of the disease-associatedprotein population from the subject's plasma.
 25. The method accordingto claim 18, wherein each ligand in the agent is selected to be specificfor an individual target protein, and to be bound by the protein with adissociation constant which is no more than 1 millimolar.
 26. A methodfor the depletion of a disease-associated protein population from theplasma of a subject in need of such treatment, which comprisesadministering to the subject a therapeutically effective amount of anon-proteinaceous agent, which agent comprises a plurality of ligandscovalently co-linked so as to form a complex with a plurality of thedisease-associated proteins in the presence thereof, wherein at leasttwo of the ligands are the same or different and are capable of beingbound by ligand binding sites present on one or more proteins selectedfrom a normal or abnormal, variant, protein of any of the followingtypes: cytokine, lipoprotein, autoantibody, acute phase protein,amyloidogenic protein, complement protein, and coagulation protein; andmonitoring the clearance of the disease-associated protein populationfrom the subject's plasma.
 27. The method according to claim 26, whereinthe ligand binding site is from an acute phase protein comprising serumamyloid A protein (SAA).
 28. The method according to claim 26, whereinthe ligand binding site is from serum amyloid P component (SAP).
 29. Themethod according to claim 26, wherein the ligand binding site is from anamyloidogenic protein selected from the group consisting of a monoclonalimmunoglobulin light chain, transthyretin, β₂-microglobulin andlysozyme.
 30. The method according to claim 29, wherein the ligandbinding site is from transthyretin and at least one of the ligandscomprises


31. The method according to claim 29, wherein the ligand binding site isfrom lysozyme and at least one of the ligands comprises a disaccharideor oligosaccharide analogue comprising N-acetylmuramic acid.
 32. Themethod according to claim 26, wherein the ligand binding site is from anautoantibody and at least one of the ligands comprises an epitope towhich the autoantibody is specific.
 33. The method according to claim 18wherein each ligand of the agent is capable of binding to the sameligand binding site.
 34. The method according to claim 18, wherein atleast two ligands of the agent are different from one another and arecapable of being bound by different proteins.
 35. The method accordingto claim 34, wherein one of the ligands of the agent is capable of beingbound by SAP.
 36. The method according to claim 31, wherein theN-acetylmuramic acid is linked to N-acetylglucosamine by a modified 1,4βglycosidic linkage, the O atom of which is replaced by a non-O atom. 37.The method according to claim 36, wherein the O atom is replaced bycarbon.
 38. The method according to claim 36, wherein the O atom isreplaced by a non-O atom that can enable hydrogen bonding contributed bythe replaced oxygen.
 39. The method according to claim 38, wherein the Oatom is replaced by nitrogen or fluorine.
 40. The method according toclaim 18, comprising clearing from the subject's plasma one or moredisease-associated proteins that are normal or abnormal, variant,proteins selected from any of the following types: cytokine,lipoprotein, autoantibody, acute phase protein, amyloidogenic protein,complement protein, and coagulation protein.
 41. The method according toclaim 40, wherein the disease-associated protein is serum amyloid Aprotein (SAA).
 42. The method according to claim 40, wherein thedisease-associated protein is serum amyloid P component (SAP).
 43. Themethod according to claim 40, wherein the disease-associated protein isan amyloidogenic protein selected from the group consisting of amonoclonal immunoglobulin light chain, transthyretin, β₂-microglobulinand lysozyme.
 44. A method for the depletion of disease-associatedproteins from the plasma of a subject in need of such treatment, whichcomprises: administering to the subject a therapeutically effectiveamount of a non-proteinaceous agent, which agent comprises a pluralityof ligands covalently co-linked to permit complexation with a pluralityof the disease-associated proteins in the presence thereof, wherein atleast two of the ligands are the same or different and are capable ofbeing bound by ligand binding sites present on the proteins; andmonitoring the clearance of the disease-associated proteins from thesubject's plasma; wherein the disease-associated proteins are one ormore normal or abnormal, variant, proteins selected from any of thefollowing types: cytokine, lipoprotein, autoantibody, acute phaseprotein, amyloidogenic protein, complement protein, and coagulationprotein.
 45. The method according to claim 44, wherein thedisease-associated protein is serum amyloid A protein (SAA).
 46. Themethod according to claim 44, wherein the disease-associated protein isserum amyloid P component (SAP).
 47. The method according to claim 44,wherein the disease-associated protein is an amyloidogenic proteinselected from the group consisting of a monoclonal immunoglobulin lightchain, transthyretin, β₂-microglobulin and lysozyme.
 48. A method forthe depletion of serum amyloid P component (SAP) from the plasma of asubject in need of such treatment, which comprises: (a) administering tothe subject a therapeutically effective amount of a non-proteinaceousagent, which agent comprises a plurality of D-proline-containing ligandscovalently co-linked to permit complexation with a plurality of SAPmolecules in the presence thereof, wherein at least two of theD-proline-containing ligands are the same or different and are capableof being bound by the calcium-dependent ligand binding sites present onthe SAP molecules; (b) binding of at least two of theD-proline-containing ligands by the ligand binding sites of SAP in theplasma; (c) forming thereby a complex between the agent and a pluralityof the SAP molecules, wherein the complex is abnormal to the subject;and (d) causing the complex to be identified by the physiologicalmechanisms of the subject and cleared from the plasma; and (e)monitoring the clearance of the SAP molecules from the subject's plasma.